Nucleic acid amplification reactions are processes by which specific nucleic acid sequences are amplified. The specific nucleic acid sequences to be amplified are referred to as target sequences. Amplification methods have become powerful tools in nucleic acid analysis and preparation. Several nucleic acid amplification methods are known. These include the polymerase chain reaction (PCR), self-sustained sequence replication (3SR), the ligase chain reaction (LCR), Q.beta. replicase amplification and strand displacement amplification (SDA). Unfortunately, the powerful ability of these nucleic acid amplification methods to amplify minute quantities of a target sequence also make them susceptible to contamination by copies of target sequences (amplicons) which may be carried over from previous amplification reactions in reagents, pipetting devices and laboratory surfaces. These contaminating products of previous amplifications may themselves be amplified in a subsequent amplification reaction. Even a few molecules of a contaminating target sequence may be readily amplified and detected, resulting in falsely positive results.
A recently developed method for inactivating contaminating amplicons in PCR involves incorporation of the nucleotide deoxyuridine triphosphate (dUTP) into amplified nucleic acid sequences in place of thymidine triphosphate (TTP). As deoxyuridine (dU) is not normally found in naturally-occurring DNA, this nucleotide serves to distinguish previously produced amplicons from new target sequences which have not yet been amplified. The uracil-containing DNAs, representing previously amplified contaminating sequences, are then treated with the enzyme uracil DNA glycosylase (UDG; also known as uracil N-glycosylase or UNG). In nature, uracil DNA glycosylase excises uracil bases from DNA which can arise as a result of either misincorporation by DNA polymerase or deamination of cytosine. For decontamination of PCR amplifications, UDG removes the intentionally incorporated uracil in amplified nucleic acid. Uracil is removed without destruction of the sugar-phosphodiester backbone, thereby producing an abasic site in the DNA. These abasic sites are susceptible to hydrolysis by heat or alkali, a process which fragments the uracil-containing DNA and renders it unamplifiable in subsequent PCR.
As employed to decontaminate PCR, a sample is treated with UDG prior to PCR amplification and the enzyme is inactivated prior to beginning the amplification reaction. This prevents removal of uracil residues from newly generated amplicons. PCR involves cycling between elevated and reduced temperatures. UDG is therefore inactivated after the decontamination treatment by incubation at high temperatures (70.degree.-80.degree. C.), a process which is compatible with the PCR. UDG is substantially inactive at the elevated temperatures used for the PCR amplification reactions themselves. However, it has been shown that upon return of the PCR sample to 4.degree.-25.degree. C. after amplification, sufficient UDG activity is still present to degrade dU-PCR amplification products. It has therefore been recommended that PCR reactions be maintained at elevated temperatures after UDG treatment (Rashtchian, A., Hartley, J. L. and Thornton, C. G., Biotechniques, volume 13, No. 2, page 180). To address the problem of residual UDG activity after heat inactivation, WO 92/01814 describes a thermolabile UDG enzyme. In a further attempt to control residual UDG activity still present after heat inactivation, Rashtchian, et al. have added a protein inhibitor of UDG (Ugi - uracil DNA glycosylase inhibitor) to PCR after heat inactivation of UDG. Ugi is a product of the bacteriophage PBS2 and inhibits host UDG to protect the phage genome during infection, as the phage substitutes dU for T during replication of its genome (Mosbaugh, D. W. and Wang, Z., Journal of Bacteriology, volume 170, No. 3 p.1082). Prior to the present invention, however, there has been no report suggesting the use of Ugi alone to inactivate UDG in the context of decontamination of nucleic acid amplification reactions.
In contrast to the PCR, several nucleic acid amplification methods are isothermal. That is, they do not involve the high/low temperature cycling of the PCR. Examples of isothermal amplification protocols are self-sustained sequence replication (3SR; J. C. Guatelli, et al. PNAS 87:1874-1878 (1990), Q.beta. replicase (P. M. Lizardi, et al. Bio/Technology 6:1197-1202 (1988), and Strand Displacement Amplification (SDA; G. T. Walker, et al. PNAS 89:392-396 (1992); G. T. Walker, et al. Nuc. Acids Res. 20:1691-1696 (1992)). Such isothermal amplification protocols present a particular problem for decontamination, as high temperature steps for inactivation of UDG may not be compatible with the reduced temperature and isothermal nature of the reaction. The SDA amplification protocol is particularly unusual in that it uses both a restriction enzyme and a polymerase to amplify DNA. DNA may be amplified by a factor of 10.sup.8 using this method. The power of the SDA system necessitates the development of a technique to insure that previously amplified material (amplicons) do not inadvertently contaminate fresh reactions. Such contamination may create falsely positive samples. The restriction enzyme most commonly used in SDA, HincII, recognizes a specific six base pair recognition sequence. SDA requires the incorporation of an .alpha.-thio derivative of deoxyadenine (dA.sub.S) into the recognition site of HincII by the polymerase in lieu of the naturally occurring dA. The mechanism of SDA is such that the SDA primers form one strand of the restriction site and the polymerase extends the primer to complete the other strand of the site using dA.sub.S TP. The dA.sub.S moiety 3' to the cut site inhibits the restriction of the modified strand. However, it does not inhibit the restriction of the unmodified strand donated by the primer.
Isothermal amplification reactions do not involve elevated temperatures as the PCR does, and it was therefore unknown prior to the present invention whether inclusion of an inhibitor of UDG alone (rather than in conjunction with heat inactivation) would be sufficient to prevent removal of uracil from the desired amplification products. Also, as the literature relating to UDG in PCR emphasizes the role of fragmentation of the abasic nucleic acids in amplicon inactivation (usually by heat), it was not previously known if removal of uracil alone would be sufficient to inactivate contaminating amplicons as templates for further amplification.
In addition to its isothermal nature, SDA differs from the PCR in several other important respects, all of which could have significant effects on the use of UDG for inactivation of contaminating amplicons. First, SDA requires nicking of the DNA by a restriction enzyme, and it has been shown that incorporation of uracil into restriction enzyme recognition sites in some cases prevents restriction. SDA also requires enzymatic displacement of the extended amplification product from the template strand by the polymerase. It was not known prior to the present invention whether 1) inclusion of uracil in the HincII restriction site and in the amplification product would prevent nicking by HincII (especially as uracil would be base paired with dA.sub.S), and/or 2) the presence of uracil or uracil base-paired with .alpha.-thio-A would prevent normal strand displacement. It was also not known whether the polymerase could successfully incorporate both unconventional nucleotides (i.e., dUTP and dA.sub.S TP) into amplification products simultaneously. In addition, the SDA KPO.sub.4 buffer system is unique in amplification reactions (PCR uses a Tris buffer) and it was not known if UDG and Ugi would be active in a KPO.sub. 4 buffer system.
In order to apply UDG decontamination to amplicons generated by SDA, it was essential that dUTP first be incorporated into amplicons (copies of a target sequence generated during the amplification reaction) via SDA. However, simple substitution of dUTP for TTP in the conventional SDA reaction (e.g., as described by Walker, et al., Nuc. Acids Res., supra) failed to produce any detectable amplification products, i.e., the conventional SDA reaction was inhibited by substitution of dUTP for TTP. Therefore, before UDG decontamination could be used in SDA reactions, the question of whether or not an SDA reaction could be developed that allowed amplification to occur in the presence of dUTP had to be addressed. It was not known whether inhibition of amplification was due to interference with restriction endonuclease nicking by incorporation of uracil into the restriction endonuclease recognition site or to some other factor(s), e.g., inability of the enzymes to incorporate dUTP or lack of displacement of dU-containing strands under the reaction conditions of conventional SDA. During the process of making the invention, it was found that incorporation of dU into the HincII restriction site does not prevent nicking by HincII, i.e., the strand which does not contain dA.sub.S is still nicked effectively. Nicking by BsoBI, in SDA reactions employing this restriction enzyme instead of HincII, is also not prevented by dU in the recognition site. It was not determined specifically which steps of the SDA reaction were inhibited in the presence of dUTP, however, by altering the reaction conditions of the conventional SDA reaction Applicants have developed a new SDA reaction in which incorporation of dU does not significantly interfere with amplification of the target sequence. Further, the KPO.sub.4 buffer typically used for SDA is compatible with UDG and Ugi activity. While magnesium can be eliminated from the decontamination reaction, it is required for the SDA reaction and for incorporation of dUTP into DNA by SDA.
By altering the reaction conditions of SDA as described herein, it has been discovered that uracil can be incorporated into the isothermally-amplified DNA without inhibition of the amplification reaction. The uracil-containing nucleic acids can then be specifically recognized and inactivated by treatment with UDG. Therefore, if dU is incorporated into isothermally-amplified DNA, any subsequent reactions can first be treated with UDG, rendering any dU containing DNA from previous amplification reactions unamplifiable. The target DNA to be amplified will not contain the dU and will not be affected by the UDG treatment. In addition, Applicants have unexpectedly found that, prior to amplification of the target, UDG can be sufficiently inhibited by treatment with Ugi alone, i.e., without the heat treatment taught in the prior art. Ugi is therefore useful in isothermal amplification reactions as a means for preventing UDG attack on new amplification products. Ugi may simply be added along with amplification enzymes to begin amplification after decontamination with UDG. These two discoveries have allowed the development of the present UDG/Ugi decontamination method for isothermal nucleic acid amplification reactions.